华西口腔医学杂志 ›› 2020, Vol. 38 ›› Issue (5): 550-557.doi: 10.7518/hxkq.2020.05.014

• 口腔肿瘤学专栏 • 上一篇    下一篇

长链非编码RNA PCGEM1通过转化生长因子β2/Smad2信号通路调控口腔鳞状细胞癌侵袭和转移的机制研究

翁旭1(), 李劲松2(), 范松2   

  1. 1. 汕头市中心医院口腔科,汕头 515031
    2. 中山大学孙逸仙纪念医院口腔科,广州 510120
  • 收稿日期:2019-12-11 修回日期:2020-06-20 出版日期:2020-10-01 发布日期:2020-10-14
  • 通讯作者: 李劲松 E-mail:wengxu2014@163.com;syong02@163.com
  • 作者简介:翁旭,副主任医师,硕士,E-mail:wengxu2014@163.com

Mechanism of long-chain noncoding RNA PCGEM1 in the regulation of the invasion and metastasis of oral squamous carcinoma cells via transforming growth factor β2/Smad2 signaling pathway

Weng Xu1(), Li Jinsong2(), Fan Song2   

  1. 1. Dept. of Stomatology, Shantou Central Hospital, Shantou 515031, China
    2. Dept. of Stomatology, Sun Yat-sen Memorial Hospital, Sun Yat-sen University, Guangzhou 510120, China
  • Received:2019-12-11 Revised:2020-06-20 Online:2020-10-01 Published:2020-10-14
  • Contact: Li Jinsong E-mail:wengxu2014@163.com;syong02@163.com

摘要: 目的 探讨长链非编码RNA(lncRNA)PCGEM1通过转化生长因子β2(TGF β2)/Smad2信号通路调控口腔鳞状细胞癌(OSCC)侵袭和转移的机制。方法 将60例OSCC患者癌组织及距离癌组织超过2 cm处的正常组织纳入研究,实时定量聚合酶链反应(qRT-PCR)检测miR-148a、lncRNA PCGEM1在OSCC组织及正常组织、人正常口腔黏膜上皮细胞(OMEC)及人源OSCC细胞株KB、BcaCD885、SCC-4、CAL27、SCC-15中的表达情况;分析lncRNA PCGEM1和miR-148a表达与患者临床病理信息之间的关系。构建lncRNA PCGEM1沉默细胞系KB-siPCGEM1及阴性对照(KB-NC),并以KB作为空白对照组,采用MTT、Transwell和划痕实验检测lncRNA PCGEM1对KB细胞增殖、侵袭和迁移能力的影响;使用生物信息学网站starBase预测lncRNA PCGEM1可以互补结合的微小RNA(miRNA),再根据www.microRNA.org网站预测相应miRNA可靶向结合的基因;免疫印迹(Western blotting)检测TGF β2/Smad2信号通路蛋白表达情况。结果 qRT-PCR结果显示,OSCC组织中lncRNA PCGEM1、miR-148a的表达水平高于正常组织(P<0.05);lncRNA PCGEM1和miR-148a在不同TNM分期、淋巴结转移和组织分化程度的患者癌组织中表达差异均有统计学意义(P<0.05);与空白对照组和KB-NC组相比,KB-siPCGEM1组细胞OD492 nm值下降,细胞侵袭数量及迁移率降低,差异均具有统计学意义(P<0.05);生物信息学预测结果显示,lncRNA PCGEM1可与miR-148a互补结合,miR-148a与TGFβ2存在靶向结合位点;qRT-PCR和Western blotting检测结果显示,KB-siPCGEM1组中miR148a、TGFβ2及p-Smad 2的表达明显低于空白对照组与KB-NC组(P<0.05),空白对照组和KB-NC组的差异无统计学意义(P>0.05)。结论 lncRNA PCGEM1在OSCC中高表达,高表达lncRNA PCGEM1可能通过上调miR-148a水平,强化TGF β2/Smad2信号通路,从而促进OSCC的进展。

关键词: 长链非编码RNA PCGEM1, 口腔鳞状细胞癌, 转化生长因子β2/Smad2信号通路;

Abstract:

Objective To investigate the mechanism underlying the regulation of the invasion and metastasis of oral squamous cell carcinoma (OSCC) by long-chain noncoding RNA (lncRNA) PCGEM1 through the transforming growth factor (TGF) β2/Smad2 signaling pathways. Methods A total of 60 OSCC cases were collected. Cancer tissues and normal tissues more than 2 cm away from cancer tissues were also collected. Real-time quantitative polymerase chain reaction (qRT-PCR) was used to detect the expression of miR-148a and lncRNA PCGEM1 in OSCC, adjacent normal tissues, oral mucosa epithelial cells, KB, BcaCD885, SCC-4, CAL27, and SCC-15. The relationship between the expression of lncRNA PCGEM1 and miR-148a and the clinicopathological information of patients was analyzed. The lncRNA PCGEM1-silenced cell line KB-siPCGEM1 and negative control (KB-NC) group were constructed, and KB was used as the blank control group. The effects of lncRNA PCGEM1 on the proliferation, invasion, and migration of KB cells were determined via MTT, Transwell, and scratch assays. The bioinformatics website starBase was used to predict the complementary binding microRNA (miRNA) of lncRNA PCGEM1. Furthermore, the genes that the miRNA could target and bind were predicted in accordance with the website www.microRNA.org. Western blotting analysis was used to detect the expression of TGF β2/Smad2 signaling pathway proteins. Results qRT-PCR results showed that the expression level of lncRNA PCGEM1 and miR-148a in OSCC tissues was higher than that in normal tissues (P<0.05). The expression of lncRNA PCGEM1 and miR-148a in the cancer tissues of patients with different TNM grades, lymph node metastasis, and tissue differentiation was statistically significant (P<0.05). Compared with those in the blank control group and the KB-NC group, OD492 nm value was significantly decreased and cell mobility was significantly reduced in the KB-siPCGEM1 group (P<0.05). Bioinformatics predictions showed that lncRNA PCGEM1 could bind to miR-148a in a complementary manner and that miR-148a had a targeted binding site with TGF β2. qRT-PCR and Western blotting analysis results showed that the expression levels of miR-148a, TGF β2, and p-Smad2 in the KB-siPCGEM1 group were significantly lower than those in the blank control and KB-NC groups (P<0.05), and no statistically significant difference between the blank control group and the KB-NC group was observed (P>0.05). Conclusion LncRNA PCGEM1 is highly expressed in OSCC. The high expression of lncRNA PCGEM1 may enhance the TGF β2/Smad2 signaling pathway by upregulating miR-148a, thus promoting the development of OSCC.

Key words: long-chain noncoding RNA PCGEM1, oral squamous cell carcinomas, transforming growth factor β2/Smad2 signaling pathway

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